Lipoproteins in living systems are classified into high-density lipoprotein (hereinafter abbreviated as HDL), low-density lipoprotein (hereinafter abbreviated as LDL), very low-density lipoprotein (hereinafter abbreviated as VLDL) and chylomicron (hereinafter abbreviated as CM) according to their specific gravity. Each class of lipoprotein has a considerably different function in vivo mainly according to the kind of apoproteins and also has a different lipid composition. It is known that, of these lipoproteins, HDL is involved in the removal of cholesterol accumulated in the cells because it receives cholesterol from tissues including arterial walls and is a risk prevention factor for various kinds of arteriosclerosis, e.g., coronary arteriosclerosis, and therefore, its level in blood is a useful index for predicting the onset of arteriosclerotic diseases.
The conventional methods for the measurement of cholesterol in HDL (hereinafter abbreviated as HDL cholesterol) consist of two operation steps, i.e., fractionation by the ultracentrifugation method, the immunochemical method, the electrophoresis method, the precipitation method, etc. and cholesterol determination. However, the operations of fractionation are complicated and time-consuming and also have a problem in respect of safety. Therefore, the measurement methods containing these separation operations are extremely inefficient and are not suited for practical use.
In recent years, various measurement methods have been proposed to solve the above problems. Examples of the methods include: a method for the fractional determination of HDL cholesterol which comprises reacting serum or plasma with cholesterol esterase and cholesterol oxidase in a buffer comprising the above enzymes, and bile salt, a bile acid derivative or dioctylsulfosuccinate, to allow cholesterol in VLDL and LDL to react with the enzymes prior to the reaction of HDL cholesterol, measuring the formed hydrogen peroxide, and then adding a nonionic surfactant having a polyoxyethylene oxide group to the reaction solution to allow HDL cholesterol to react with the enzymes (see patent document No. 1); and a method for the measurement of HDL cholesterol which comprises reacting serum with pancreas-derived cholesterol esterase and cholesterol oxidase in a buffer comprising the enzymes, a surfactant belonging to the group of bile acids and a nonionic surfactant at a specific pH and a specific temperature (see patent document No. 2). In the method described in patent document No. 2, the reaction of LDL cholesterol with the enzymes proceeds first and then the reaction of HDL cholesterol with the enzymes proceeds, which enables the measurement of HDL cholesterol. However, these methods require a lot of time for measuring and are not always specific for the measurement of HDL cholesterol.
Known examples of the methods for the measurement of HDL cholesterol by aggregating lipoproteins other than HDL include: a method using a reagent for aggregating lipoproteins other than HDL (e.g., dextran sulfate), a divalent metal salt and a chemically modified enzyme (see patent document No. 3); a method using a reagent which forms a complex with lipoproteins other than HDL (e.g., polyanion) and a surfactant which does not dissolve lipoproteins (e.g., polyoxyethylene-polyoxypropylene copolymer) (see patent document No. 4); a method using a polyanion (e.g., dextran sulfate), a divalent metal salt, a specific nonionic surfactant and albumin which is different from the albumin contained in a sample (see patent document No. 5); and a method for the measurement of HDL cholesterol in serum or plasma which comprises treating serum or plasma with a solution containing a lipoprotein fractionating agent (a combination of a polyanion such as dextran sulfate and a divalent cation such as magnesium ion), reacting the obtained mixture with cholesterol esterase and cholesterol oxidase in the presence of an anionic surfactant (alkylsulfonic acid, bile acid or its derivative) without subjecting the mixture to solid-liquid separation, and measuring the formed hydrogen peroxide (see patent document No. 6).
These methods for the measurement of HDL cholesterol by aggregating lipoproteins other than HDL have a good correlation with conventional standard methods. However, there are problems with these methods such as inaccuracy due to turbidity caused by aggregates formed by the reaction, and an excessive load to an autoanalyzer due to deposition of metal hydroxide formed by the reaction with metal salt in a reaction solution when reaction cells are washed with an alkali solution.
Known examples of the methods for the measurement of HDL cholesterol without aggregating lipoproteins other than HDL include: a method for the measurement of HDL cholesterol in a biological sample which comprises reacting the biological sample with pancreas-derived cholesterol esterase and cholesterol oxidase in the presence of bile acid or its salt and albumin, and measuring a compound consumed or formed by the enzymatic reaction (see patent document No. 7); and a method for the measurement of HDL cholesterol in a sample which comprises reacting the sample with lipoprotein lipase which preferentially acts on HDL fraction and/or cholesterol esterase and cholesterol oxidase in the presence of a nonionic surfactant with an HLB value of 16 or more which has reaction selectivity to the HDL fraction (see patent document No. 8). Also known is a method in which cholesterol in lipoproteins other than HDL is preferentially converted into hydrogen peroxide with acyl polyoxyethylene sorbitan ester, and after the formed hydrogen peroxide is eliminated, HDL cholesterol is enzymatically measured by adding polyoxyethylene alkyl ether (see patent document No. 9).
However, these methods for the measurement of HDL cholesterol without aggregating lipoproteins other than HDL sometimes have the problem of inaccuracy of measurement values due to incomplete elimination of cholesterol in lipoproteins other than HDL and non-specific reaction with cholesterol in lipoproteins other than HDL.
Additional known examples of the methods for the measurement of HDL cholesterol without aggregating lipoproteins other than HDL include: a method for the measurement of HDL cholesterol in a sample which comprises reacting the sample with i) cholesterol esterase and cholesterol oxidase or ii) cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase in an aqueous medium comprising a nonionic surfactant, a polyanion and albumin, and measuring the formed hydrogen peroxide or reduced coenzyme (see patent document No. 10); and a method for the measurement of HDL cholesterol which comprises reacting a sample with cholesterol esterase and cholesterol oxidase, or cholesterol esterase, oxidized coenzyme and cholesterol dehydrogenase in an aqueous medium comprising a bile acid derivative, and measuring the formed hydrogen peroxide or reduced coenzyme (see patent document No. 11).
Patent Document No. 1:
                Japanese Published Unexamined Patent Application No. 69999/87Patent Document No. 2:        Japanese Published Unexamined Patent Application No. 126498/88Patent Document No. 3:        Japanese Published Unexamined Patent Application No. 131197/96Patent Document No. 4:        Japanese Published Unexamined Patent Application No. 201393/96Patent Document No. 5:        Japanese Published Unexamined Patent Application No. 285298/97Patent Document No. 6:        Japanese Published Unexamined Patent Application No. 116996/96Patent Document No. 7:        WO97/40376 pamphletPatent Document No. 8:        WO00/52480 pamphletPatent Document No. 9:        Japanese Published Unexamined Patent Application No. 299/97Patent Document No. 10:        WO04/035816 pamphletPatent Document No. 11:        WO04/035817 pamphlet        